Purification and sterilization processes applied to protein preparations may result in partial denaturation of the protein employed. Hitherto it was preferable, particularly based on economic considerations, to separate out and discard denatured protein. Protein prepared by gene manipulation in prokaryotes is largely in a biologically inactive form.
In order to raise the yield of "natural" protein, that is to say that with the correct spatial structure and the biological activity of the natural protein, it is necessary first for the polypeptide chain to be unfolded to give a random coil, and any incorrect disulfide bridges which are present to be reduced. This is normally carried out by incubation in at least 4 mol/l guanidine hydrochloride solution or at least 6 mol/l urea solution, where appropriate with the addition of a reducing agent such as dithiothreitol (DTT). Subsequently, the formation of the correct protein structure has, to date, been brought about by dilution (at least 1:40) or dialysis against a "physiological" buffer solution.
It is hardly possible to use either method industrially. Dilution of volumes which are large at the outset, followed by reconcentration, is time-consuming, troublesome and costly. This is similarly true of dialysis of large volumes. Furthermore, slow removal of denaturing agent considerably reduces the reactivation yield because side-reactions, such as aggregations, take place preferentially in the intermediate range of concentrations of denaturing agent.